Microfluidic devices for isolating particles

ABSTRACT

In one aspect, a system for isolating particles includes a first array of magnets, a second array of magnets arranged generally in parallel with and spaced apart from the first array of magnets, and a microfluidic device. The microfluidic device includes a substrate, an inlet arranged on the substrate and configured to receive a fluid sample, an outlet arranged on the substrate, a first region of the substrate including a channel connected to the inlet, where the first region of the substrate is arranged to sandwich the channel between the first and second arrays of magnets, and a second region of the substrate in fluid communication with the channel and including a particle capture zone containing a plurality of particle capture sites, where each particle capture site including a receptacle sized to confine a first type of particle and an opening in fluid communication with the receptacle, wherein a size of the receptacle is larger than a size of the opening.

RELATED APPLICATIONS

The present application claims priority from provisional U.S.Provisional Patent Application Ser. No. 61/917,268, filed Dec. 17, 2013,the subject matter of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

This disclosure relates to microfluidic devices for isolating particles.

BACKGROUND

Magnetic fields can have broad applications in biotechnology andmedicine. For example, systems utilizing magnetic fields can haveapplications in cancer diagnostics, drug discovery, and stem cellresearch, among others. One particular area includes magnetic separationof cells, in which cells of interest are attached to magnetic biomarkersin a solution and the solution is then introduced into an area having amagnetic field. The magnetic field serves to isolate and/or filter thecells having the attached biomarkers for subsequent analysis,modification, or use.

SUMMARY

This disclosure describes techniques and systems for utilizing arrays ofmagnets to isolate target particles (e.g., rare target particles such asrare cells, e.g., circulating tumor cells or fetal cells in maternalblood) from other particles (e.g., other types of cells) in a samplefluid (e.g., biological fluid, such as blood sample). For example, thearrays of magnets can be assembled onto a microfluidic device togenerate strong magnetic fields in regions of one or more channels inthe microfluidic device. The strong magnetic fields can isolateparticles bound to magnetic beads from other particles in the samplefluid. The design and arrangement of the arrays of magnets canefficiently isolate particles while allowing high throughput of samplefluid in the microfluidic device.

In some embodiments, the techniques and systems disclosed herein enablenegative and positive selection and separation of particles in a samplefluid as the fluid flows through a microfluidic device by utilizingarrays of magnets and size-based particle capture. For example, thearrays can be arranged as one-dimensional or two-dimensional“checkerboards” of magnets with alternating polarities, as described infurther detail below. The arrays of magnets can be used toimmuno-magnetically deplete abundant host cells (e.g., leukocytes).Following negative selection, remaining particles (e.g., target cancercells or fetal cells) that are not immuno-magnetically depleted can beflowed into a particle capture zone for capturing and sorting accordingto their size. For example, the captured particles can be manipulatedusing a tweezer device (e.g., optical or acoustic tweezer device) andanalyzed in an efficient manner. For example, the captured particles canbe analyzed in situ for a comprehensive and multifaceted evaluation,including single cell enumeration and imaging, molecular and geneticprofiling, and drug-treatment responses. In particular, a user orautomated system can utilize the tweezer device to lift and displacecaptured particles for analysis.

In one aspect, a system for isolating particles includes a first arrayof magnets, a second array of magnets arranged generally in parallelwith and spaced apart from the first array of magnets, and amicrofluidic device. The microfluidic device includes a substrate, aninlet arranged on the substrate and configured to receive a fluidsample, an outlet arranged on the substrate, a first region of thesubstrate including a channel connected to the inlet, where the firstregion of the substrate is arranged to sandwich the channel between thefirst and second arrays of magnets, and a second region of the substratein fluid communication with the channel and including a particle capturezone containing a plurality of particle capture sites, where eachparticle capture site including a receptacle sized to confine a firsttype of particle and an opening in fluid communication with thereceptacle, wherein a size of the receptacle is larger than a size ofthe opening.

Implementations of this aspect may include or more of the followingfeatures.

In some implementations, the first array of magnets and the second arrayof magnets can each include magnets arranged such that adjacent magnetshave dipole moments aligned in opposite directions.

In some implementations, the magnets in the first array of magnets andthe second array of magnets can each include NdFeB, SmCo, Fe, Ni, Co,FePt, MnFe2O4, CoFe2O4, NiFe2O4, ZnMnFe2O4, or iron oxide.

In some implementations, a length of at least one of magnets in thefirst array of magnets along a fluid propagation direction through thechannel can be 100 μm or more.

In some implementations, a distance between the first array of magnetsand the second array of magnets can be 0.5 mm or more.

In some implementations, a peak magnitude value of a magnetic fieldstrength at a point between the first array of magnets and the secondarray of magnets can be about 0.45 T or more.

In some implementations, a magnitude of an average magnetic fieldstrength along a line extending from the first array of magnets to thesecond array of magnets can be about 0.35 T or more.

In some implementations, the system can further include a tweezer deviceconfigured to displace the particle captured in one of the particlecapture sites. The system can further include a receiver deviceconfigured to receive the displaced particle. The tweezer device can bean optical tweezer device. The optical tweezer device can include anoptical source for generating an optical beam, and a lens for focusingthe optical beam into one of the particle capture sites.

In some implementations, for at least one particle capture site, thereceptacle can be sized to receive and confine a single first type ofparticle, and, for the at least one particle capture site, the openingcan be small enough to prohibit passage of the first type of particleand large enough to allow passage of a second type of particle.

In some implementations, at least one particular capture site caninclude a first wall and a corresponding second wall, where thereceptacle bounded by the first and second wall, and where the openingis defined between an end of the first wall and an end of the secondwall.

In some implementations, at least one of the particle capture sites caninclude a wall, where the receptacle is defined by a recess in the wall,and wherein the opening extends from the receptacle on a first side ofthe wall to a second opposite side of the wall.

In some implementations, during operation of the system the receptacleand opening of each particle capture site can be aligned substantiallyparallel to a direction of fluid flow in the second region.

In some implementations, the particle capture sites can be arranged inat least two rows, where the particle capture sites in each row arespatially offset from the particle capture sites in an adjacent row intwo orthogonal directions.

In some implementations, the system can further include at least oneadditional inlet in fluid communication with the second region, wherethe additional inlet is arranged to receive one or more additional fluidsamples and output the one or more additional fluid samples through atleast the second region.

In some implementations, the system can further include a fluid manifoldarranged on the substrate between the particle capture zone and theoutlet.

In another aspect, a method for isolating particles includes providing afirst and a second array of magnets, where the first and second arraysof magnets are positioned to sandwich a region including a channel of amicrofluidic device. The method also includes providing a sample fluidcomprising a plurality of particles into the channel, where at least onefirst type of particle is bound to a magnetic bead, and where a magneticfield extending between the first and second magnet arrays within thechannel causes the at least one first type of particle to separate fromremaining particles in the fluid sample. The method also includesproviding the fluid sample containing the remaining particles to aparticle capture zone of the microfluidic device, where the particlecapture zone comprises a plurality of particle capture sites. The methodalso includes capturing one or more second type of particles at theparticle capture sites of the microfluidic device, where the one or moresecond type of particles are not bound to a magnetic bead. Each particlecapture site includes a receptacle sized to confine the second type ofparticle and an opening in fluid communication with the receptacle, andwhere a size of the receptacle is larger than a size of the opening.

Implementations of this aspect may include or more of the followingfeatures.

In some implementations, capturing the second type of particle caninclude receiving a single second type of particle in a receptacle ofone of the particle capture sites.

In some implementations, the method can further include displacing theone or more second type of particles from the particle capture sitesusing an optical tweezer. Displacing the one or more second type ofparticles from the particle capture sites can include displacing asingle one of the second type of particles from a single one of theparticle capture sites. The optical tweezer can be configured to providea plurality of optical traps.

In some implementations, the method can further include collecting thedisplaced second type of particle in a receiver device.

In some implementations, the method can further include flowing a firstadditional fluid through at least the particle capture zone. The firstadditional fluid can include a plurality of first fluorescent markers,and the method can further include allowing the plurality of the firstfluorescent markers to bind to one or more of the second type ofparticle. The method can further include optically exciting the firstfluorescent markers bound to the one or more second type of particle,obtaining an image of the one or more second type of particle, anddetermining a characteristic of the second type of particle based on theobtained image. The characteristic can include a presence or absence ofa first biomarker expressed by the second type of particle. Determiningthe characteristic of the second type of particle based on the obtainedimage can include determining an intensity of fluorescence associatedwith the second type of particle based on the obtained image.

In some implementations, the method can further include flowing anelutant through at least the particle capture zone, where flowing theelutant causes the fluorescently labeled particles to release fromsecond type of particle. The method can further include flowing a secondadditional fluid sample through at least the particle capture zone,where the second additional fluid includes a plurality of a second typeof fluorescent marker, and where one of more of the second type offluorescent markers bind to one or more of the second type of particle.

In some implementations, the method can further include opticallyexciting one or more of the second fluorescent markers bound to secondtype of particle, obtaining a second image of the second type ofparticle, and determining a characteristic of the second type ofparticle based on the obtained second image. The characteristic caninclude a presence or absence of a second biomarker expressed by thesecond type of particle different than the first biomarker. Determiningthe characteristic of the second type of particle based on the obtainedsecond image can include determining an intensity of fluorescenceassociated with the second type of particle based on the obtained secondimage.

In some implementations, the first additional fluid substance caninclude a drug such that the second type of particle is exposed to thedrug. A concentration of the drug within the additional fluid can begraded.

In some implementations, the method can further include culturing the atleast one second type of particle in the particle capture zone after theat least one second type of particle is captured.

The techniques and systems disclosed in this specification providenumerous benefits and advantages (some of which can be achieved only insome of the various aspect and implementations) including the following.The disclosed systems can be assembled as modular components, which canlead to ease and cost effective manufacturing. For example,manufacturing of separate modular components can be easier and lessexpensive than fabricating an integrated system. Moreover, the approachusing modular components can allow a user to easily replace differentsample fluids, magnets, channel types so as to increase throughput ofthe analysis while maintaining a sealed system without fluid leakage.The systems can include magnetically detachable fluidic ports, which cansignificantly simplify the fluidic connection to microfluidic devices.

In general, the disclosed techniques can be used to combine bothnegative and the positive selection of particles (e.g., negative andpositive cell enrichment), thereby enabling high throughput andnon-biased isolation of target particles. Arrays of magnets can be usedto generate a strong magnetic force with a relatively long range, andthereby allowing efficient isolation of particles attached to magneticbeads. Furthermore, because the magnetic force can be long range, thearrays of magnets can be placed outside a microfluidic device so thatthe magnets do not contact the sample fluid. Thus, contamination of themagnets can be avoided, and the magnets may easily be reused for testingof different sample fluids.

In general, the disclosed techniques can be used to provide systems withparticle capture sites with a hydrodynamic design to ensure asingle-cell trapping per site, while minimizing the risk of clogging themicrofluidic devices. The particle capture sites can be further laid outinto a size-gradient array, so as to differentially capture particlesbased on their size. Furthermore, the systems can include a tweezerdevice to manipulate particles captured in particle capture sites. Forexample, the tweezer device can be used to manipulate and retrievecaptured cells at a single-cell resolution. This capability, combinedwith emerging molecular techniques, can enable the study of single-cellgenomics and proteomics.

In general, the disclosed techniques also enable analysis of individualrare cells through the combination of magnetic depletion of largequantities of undesired cells and capture sites configured to receiveand trap individual rare cells. The capture sites have the furtheradvantage of being able to provide additional filtering so that onlyrare cells are trapped and analyzed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present implementations, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages will be apparent from the followingdetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a system for isolating particles.

FIG. 2 is a schematic diagram showing a portion of the system describedin FIG. 1.

FIG. 3 is a schematic diagram showing a portion of an array of magnets.

FIG. 4 is a schematic diagram showing a portion of a cross-section ofthe system described in relation to FIG. 2.

FIG. 5A is a schematic diagram showing an example of a microfluidicdevice.

FIG. 5B is a schematic diagram showing another example of a microfluidicdevice.

FIG. 6A is a schematic diagram showing a portion of the microfluidicdevice described in FIG. 5A.

FIG. 6B is a schematic diagram showing a top view of an example of aparticle capture zone.

FIG. 6C is a schematic diagram showing another example of a particlecapture zone.

FIG. 7A is a schematic diagram showing an example of a tweezer device.

FIG. 7B is a schematic diagram showing another example of a tweezerdevice.

FIG. 8 is a schematic diagram showing a top view of the particle capturezone described in relation to FIG. 6B.

FIGS. 9A-9C are schematic diagrams of images showing a process formanipulating a target particle.

FIG. 10 is a schematic diagram showing a portion of the system describedin FIG. 1.

FIG. 11 is a flow chart depicting exemplary operations for isolatingparticles.

FIG. 12 is a schematic diagram showing an example of a controller.

FIGS. 13A and 13B are schematic diagrams showing two configurations ofarrays of magnets and their calculated magnetic field distribution.

FIG. 14 is a plot depicting calculated magnitude of magnetic field(|B{right arrow over (|)}).

FIG. 15 is a plot depicting calculated magnitude of magnetic force|{right arrow over (F)}_(B)| of a spherical particle.

FIG. 16 is a schematic diagram of a portion of a microfluidic deviceused in a measurement.

FIG. 17 is a plot depicting measurement results of enrichment ratios.

FIGS. 18A-C are images showing a particle capture site in a microfluidicdevice with an optical trap.

FIG. 19 is an image showing an example of a particle capture zone.

FIG. 20 is a schematic diagram illustrating a portion of an example of acapture zone.

FIG. 21A is an image showing an overlay of four imaging channels after amixture of DB cells (a B lymphoblast cell line) and Daudi cells (a Blymphoblast cell line) was captured and stained on a microfluidicdevice.

FIG. 21B is an image showing selected portions of the overlay shown inFIG. 21A.

FIG. 22A is an image depicting the intensity of light within thephycoerythrin (PE) channel for a portion of a capture region.

FIG. 22B is an image that results after performing an intensity-basedthresholding on the image shown in FIG. 22A.

FIG. 22C is an image showing masks that are generated based on the imageshown in FIG. 22B.

FIG. 22D is an image showing the application of the masks shown in FIG.22C to additional images.

FIG. 23 is a collection of plots depicting the distribution of maskedregions of images of a capture zone on the basis of a first intensity(lambda, k) and a second intensity (kappa, κ) for different cells.

FIG. 24 is an image depicting fluorescently labeled Rec-1 cells andJurkat cells.

FIGS. 25A-C are diagrams showing an example drug screening process.

FIGS. 26A-D are diagrams showing an example particle cycling process.

DETAILED DESCRIPTION

The methods and systems described herein can be implemented in manyways. Some useful implementations are described below. The scope of thepresent disclosure is not limited to the detailed implementationsdescribed in this section, but is described in broader terms in theclaims.

Introduction

Various particles (e.g., tumor cells) are important biomarkers forclinical practice as well as fundamental research. For example,circulating tumor cells (“CTCs”), which are shed from primary tumors,can be a harbinger of tumor expansion. Proteomic characterization ofproliferative markers such as Ki-67 and hormonal markers such asandrogen receptor in prostate cancer can be predictive of treatmentoutcomes. To detect and characterize particles such as tumor cells andother biomarkers, a user often flows a sample fluid (e.g., a biologicalfluid such as a blood, e.g., whole blood or buffy coat, lymph,cerebrospinal fluid, urine, saliva, or a sputum sample) through channelsof a microfluidic device to separate different types of particles.Subsequent characterization is based on the separation of particles.However, routine detection and characterization of particles in thesample fluid typically require screening a large number of particles andenrichment of heterogeneous targets to obtain accurate results.

Accordingly, the techniques described herein provide systems that can beused to isolate target particles with high enrichment while reducing theprobability of missing the detection of a rare target particle in agiven sample fluid. For example, the systems can utilize arrays ofmagnets sandwiching regions of channels of the microfluidic device,through which the sample fluid flows. In such regions of the channels,the design and arrangement of the arrays of magnets can provide strongmagnetic forces onto particles bound to magnetic beads so that suchparticles are attracted towards the magnets, and thereby isolating theattracted particles from remaining particles in the sample fluid. Forexample, abundant host particles (e.g., blood cells) can be magneticallydepleted in this manner so that target cells that are not magneticallydepleted can be enriched with high efficiency and throughput. Moreover,the disclosed arrangements of magnet arrays can provide a strongermagnetic force than the case in which magnets are positioned on only oneside of the channels.

Moreover, the microfluidic devices can include a particle capture zonecontaining particle capture sites. These sites can be used to captureparticles with specific sizes among the remaining particles. Forexample, the size and shape of different sites can be designed tocapture specific particles while allowing other particles to passthrough. In this approach, the microfluidic devices can provide positiveselection of particles along with a size sorting capability.

Both negative and positive selection can be enabled by a singlemicrofluidic device. Thus, the microfluidic device can provide highthroughput of separating particles so that a user may carry out testseasily in a fast manner, which may allow ease of repeated measurementsas well as operation of different types of measurements. Detailedembodiments and various advantages are described in the following.

General Systems

FIG. 1 is a schematic diagram showing a system 100 for isolatingparticles. System 100 can include an array of magnets 102 and an arrayof magnets 104 sandwiching a region of a microfluidic device 110 so asto provide magnetic fields in the region. The microfluidic device 110can be coupled to a sample fluid provider 112 so that the microfluidicdevice 100 can receive sample fluid from the sample provider 112. Insome embodiments, coupling 111 can be a tube connecting the sample fluidprovider 112 to an inlet (not shown) of the microfluidic device 110. Thesample fluid received through the inlet can flow through channels (notshown) of the microfluidic device towards an outlet (not shown) of themicrofluidic device 100. During the flow, the magnetic fields providedby the arrays of magnets 102 and 104 isolate particles bounded withmagnetic beads. Thus, particles bounded to magnetic beads becomeattached to walls of the channels by the magnetic forces and suchparticles are thus separated from other particles in the sample fluid.On the other hand, particles that are not attracted by the magneticforces can flow towards the outlet.

As described later in detail, the microfluidic device 110 can include aparticle capture zone for capturing particles in the sample fluid. Thesystem 100 can include a tweezer (e.g., optical tweezer, acoustictweezer) device 120 coupled to the microfluidic device 110 throughcoupling 121. In some embodiments, the coupling 121 can be an opticalbeam used to trap particles captured in the particle capture zone. Forexample, the tweezer device 110 can lift and move a specific capturedparticle (selected either by an operator of the system who visualizesthe particles on a monitor, or by a system that automatically targetsspecific particles, e.g., based on a fluorescent or other reportergroup). When the specific captured particle is lifted and the tweezerdevice 120 releases the lifted particle, fluid flow can transport thelifted particle towards the outlet.

The outlet of the microfluidic device 110 can be coupled to a receiverdevice 114. In some embodiments, coupling 115 can be a tube connectingthe outlet of the microfluidic device 110 to the receiver device 114.The receiver device 114 can have multiple containers for containingselected particles or fluids. For example, in one container, thereceiver device 114 can contain particles that are not isolated by themagnetic forces or captured in the particle capture zone. In anothercontainer, the receiver device 114 can contain selected particles thatare specifically released by the tweezer device 120 and transported tothe outlet.

FIG. 2 is a schematic diagram showing a portion of the system 100described in FIG. 1. Separations between various elements are depictedschematically for illustrative purposes. Global coordinate 290 indicatesthe orientation of the schematic drawing. In this example, themicrofluidic device 110 has a substrate, which includes a microfluidicchannel layer 204 and a plate 202. The plate 202 supports themicrofluidic channel layer 204. In some embodiments, the plate 202 caninclude glass, microslide, or plastic material. The plate 202 can betransparent to visible light for imaging or optical manipulationpurposes. The microfluidic channel layer 204 can include apolydimethylsiloxane (PDMS), glass, or plastic layer. In certainembodiments, either of the microfluidic channel layer 204 and plate 202can be a plastic mold configured to hold either or both of the arrays ofmagnets 102 and 104.

The microfluidic channel layer 204 can include inlet 502, channel 506,particle capture zone 508, fluid manifold 505 and outlet 504, which willbe described later. In this example, another plate (not shown) ispositioned between the array of magnets 102 and the microfluidic channel204. Such configuration is described later in relation to FIG. 4. Insome embodiments, either of the arrays of magnets 102 or 104 can bedirectly in contact with the microfluidic channel 204 without a plate inbetween. The channel 506 can be embedded within microfluidic channellayer 204 so that top and bottom surfaces of the microfluidic channellayer 204 is in contact with either the arrays of magnets 102 and 104and the plates, respectively. In certain embodiments, channel 506 can beopen on one side of the microfluidic channel layer 204 so that channelsare open facing towards the plate 202. Thus, the plate 202 can act as asealing layer for the channels in the microfluidic channel layer 204.

In some embodiments, the system 100 can include detachable inletelements 210 and 212. For example, the detachable inlet elements 210 and212 can be cylindrical magnets with a fluidic port of a hollow bore. Theinlet elements 210 and 212 can clamp and hold the microfluidic channellayer 204 and the plate 202. In this case, the inlet elements 210 and212 can clamp and seal the microfluidic device 110 with mitigatedeffects of stress applied to the microfluidic device 110 due toclamping. The inlet elements 210 and 212 can also allow connection oftubes to the sample provider 112. In certain embodiments, the inletelements 210 and 212 can function as a stand-alone reservoir of samplefluid. Because the magnetic force between the inlet elements 210 and 212can tightly clamp the microfluidic device 110, the inlet elements 210and 212 can effectively seal the tube connection between the sampleprovider 112 and the microfluidic device 110 without fluid leakage. Onthe other hand, a user may easily remove the inlet elements 210 and 212,for example, in a direction orthogonal to the magnetic forces betweenthe inlet elements 210 and 212. Thus, the user may easily replace onemicrofluidic device from another without issues of fluid leakage.

The system 100 can include detachable outlet elements 220 and 222.Similarly, as described in relation to inlet elements 210 and 212, theoutlet elements 220 and 222 can be magnetic and be used to clamp themicrofluidic device 110 for sealing while allow easy replacement of themicrofluidic device.

In certain embodiments, either of the detachable inlet and outletelements 210, 212, 220 and 222 can be screwed onto the microfluidicdevice 110. This approach can provide a tight seal between themicrofluidic device 110, sample provider 112, and receiver device 114.

In some embodiments, the microfluidic device 110 can include anotherplate (not shown) between the microfluidic channel layer 204 and thearray of magnets 102. In this approach, the microfluidic channel layer204 is sandwiched between the plate 202 and another plate, which canlead to uniform pressure being applied over the microfluidic channellayer 204.

The array of magnets 102 is positioned adjacent to the microfluidicdevice 110 in the positive z-direction. The array of magnets 104 ispositioned adjacent to the microfluidic device 110 in the negativez-direction. Thus, in this example, the arrays of magnets 102 and 104sandwich and provide magnetic fields in a region of the microfluidicdevice 110. The magnetic attraction between the arrays of magnets 102and 104 can clamp the microfluidic device 110 so as to provide a tightseal without leakage. In certain embodiments, either of the arrays ofmagnets 102 and 104 can be fixed in a molding of the microfluidic device110 so that either of the arrays can hold onto the microfluidic device110 without the presence of the other array.

Moreover, magnetic dipole moments of the magnets in the arrays ofmagnets 102 and 104 are arranged to provide a strong magnetic fieldstrength and large magnetic field gradient in the region of the one ormore channels in the microfluidic device 110. Orientations of themagnetic dipole moments of the magnets are described below.

Referring back to FIG. 2, a zoomed view depicts a 1-dimensional (1D)subset array 230 of the array of magnets 102 including five magnets231-235. Another zoomed view depicts a 1D subset array 250 of the arrayof magnets 104 including five magnets 251-255. Only five magnets aredescribed for illustrative purposes according to global coordinate 291.

In this disclosure, two magnets being “directly adjacent” to one anothermeans that the two magnets are positioned next to one another withoutanother magnet in between. In this example, the magnet 231 has amagnetic dipole moment 236 pointing in the z-direction. Magnet 232,which is directly adjacent to magnet 231, has a magnetic dipole moment237 pointing in the negative z-direction. Magnet 233, which is directlyadjacent to magnet 232 and 234, has a magnetic dipole moment 238pointing in the positive z-direction. Magnet 234 has its magnetic dipolemoment 239 pointing in the negative z-direction, and magnet 235 has itsmagnetic dipole moment 240 pointing in the positive z-direction.

Furthermore, magnets 251, 253 and 255 of subset array 250 have theirrespective magnetic dipole moments 256, 258 and 260 pointing in thepositive z-direction. Magnets 252 and 254 have their respective magneticdipole moments 257 and 259 pointing in the negative z-direction.

Generally, each of the arrays of magnets 102 and 104 can include anumber of magnets to cover the area of channels 506 in the microfluidicdevice 110. For example, the array of magnets 102 can include 5 or more(e.g., 10 or more, 30 or more, 50 or more, 100 or more, 500 or more)rows. Each row of the array of magnets 102 can include 5 or more (e.g.,10 or more, 30 or more, 50 or more, 100 or more, 500 or more) magnets.In some embodiments, a length of the whole array of magnets 102 can be30 mm or more (e.g., 40 mm or more, 50 mm or more).

In the example shown in FIG. 2, magnet 231 has a length 289 of 1 mm. Insome embodiments, the length 289 can be 0.1 mm or more (e.g., 0.3 mm ormore, 0.5 mm or more, 1.5 mm or more, 2 mm or more, 3 mm or more). Insome cases, the length 289 can be between 0.1 mm and 10 mm (e.g.,between 0.1 mm and 1 mm, between 1 mm and 2 mm, between 2 mm and 3 mm,between 3 mm and 4 mm, between 4 mm and 5 mm, or between 5 mm and 10mm). The other magnets can have the same length as length 289. Incertain embodiments, the length 289 can have a maximum length of about 5mm or less (e.g., 4 mm or less, 3 mm or less). Generally, differentmagnets can have the same or different lengths and the magnets can bearranged to provide a desired magnetic field distribution according tothe design of channel 506.

It is understood that the array of magnets 104 can have similarcharacteristics described in relation to the array of magnets 102described in the preceding paragraphs.

The individual magnets can be permanent magnets. In some embodiments,the magnets can comprise a magnetic material including NdFeB, SmCo, Fe,Ni, Co, FePt, MnFe₂O₄, CoFe₂O₄, NiFe₂O₄, ZnMnFe₂O₄, or iron oxide. Themagnetic material can be selected to have a high magnetic permeabilityfor providing strong magnetic fields.

FIG. 3 is a schematic diagram showing a portion of the array of magnets102 in a different perspective according to global coordinate 292. Inthis disclosure, the “dot” notation refers to a direction pointing outof the drawing plane, and the “cross” notation refers to a directionpointing into the drawing plane. Accordingly, FIG. 3 depicts magnet 231with its magnetic dipole moment 236 pointing in the positive z-directionand magnet 234 with its magnetic dipole moment 239 pointing in thenegative z-direction. As mentioned earlier, magnet 232, which isdirectly adjacent to magnets 231 and 233, has its magnetic dipole moment237 pointing in the negative z-direction. Thus, in the example shown inFIG. 3, magnets directly adjacent one another have magnetic dipolemoments pointing in different direction. Further, FIG. 3 depicts theorientation of magnetic dipole moments of the magnets arranged in a 2Darray in a plane parallel to the x- and y-direction. It is understoodthat the magnetic dipole moments of the magnets are arranged in analternating manner, such that adjacent magnets have dipole momentsaligned in opposite directions. Such a configuration of array of magnets102 described in relation to FIG. 3 may also be referred as a“checkerboard” pattern.

The arrays of magnets 104 lie below the arrays of magnets 102 in thenegative z-direction, as shown in FIG. 2. As similarly described inrelation to FIG. 3, subset array 250 includes magnets 251-255 which havemagnetic dipole moments 256-260, respectively, pointing in differentdirections for directly adjacent magnets. It is understood that themagnetic dipole moments of the magnets are arranged in an alternatingmanner.

Referring back to FIG. 2, the array of magnets 102 lie in a first planeparallel to the x-y plane. The array of magnets 104 lie in a secondplane parallel to the x-y plane. Hence, in this example, the first andsecond planes are parallel to each another. In some embodiments, thefirst and second planes are substantially parallel to each anotherwithin 10° (e.g., within 5°, within 3°, within 1°).

The arrays of magnets 102 and 104 are arranged to sandwich themicrofluidic device 110. Thus, the array of magnets 102 is displacedrelative to the array of magnets 104 in the positive z-direction, whichare generally parallel to each other in some embodiments. In otherembodiments, the two arrays may be arranged at an angle, e.g., a slightangle, to each other. The relative positions between individual magnetsof the arrays of magnets 102 and 104 are described in comparison tosubset arrays 230 and 250 in FIG. 2. The magnet 231 is positionedrelative to magnet 251 in the positive z-direction, and magnet 232 ispositioned relative to magnet 242 in the positive z-direction.Similarly, magnets 233-235 are positioned relative to magnets 253-255 inthe positive z-direction. Generally, magnets of the array of magnets 102are positive relative to magnets in the array of magnets 104 in thepositive z-direction so that magnets with magnetic dipole moments pointin the same direction are aligned to one another. For example, magneticdipole moment 236 is aligned to its corresponding magnetic dipole moment256, and the magnetic dipole moment 237 is aligned to its correspondingmagnetic dipole moment 257. In certain embodiments, at least some of themagnetic dipole moments and corresponding magnetic dipole moments canpoint substantially in the same direction. For example, at least some ofthe magnetic dipole moments and corresponding magnetic dipole momentspoint in the same direction within 1° or less (e.g., 2° or less, 5° orless, 10° or less).

To further illustrate the arrangement of the arrays of magnets 102 and104, FIG. 4 is a schematic diagram showing a portion of a cross-sectionof the system 100 described in relation to FIG. 2. Global coordinate 291indicates the perspective of the cross-section. For illustrativepurposes, magnets 231, 231, 251 and 252 are depicted with theirrespective magnetic poles 402 and 404. The magnet 231 is position abovethe magnet 251 in the positive z-direction, and the magnet 232 ispositioned above the magnet 252 in the positive z-direction. As shown,magnetic dipole moments 231 and 251 point in the same direction, andmagnetic dipole moments 232 and 252 point in the same direction,opposite of that of magnetic dipole moments 231 and 251. As shown inFIG. 3, alternating arrangement of the magnetic dipole moments extend inboth the x- and y-directions. Such a configuration can generate strongmagnetic fields 410 and larger magnetic field gradients within channel506 of the microfluidic device 110.

The alignment of magnetic dipole moments 231 and 251 reinforce themagnetic fields of the magnetic dipole moments 231 and 251 to providestrong magnetic fields. Such magnetic fields at certain locations can beeven stronger than that of the case when only one array of magnetsexists adjacent to the channel 506. Moreover, the alternating magneticdipole moments above and below the channels lead to a large gradient ofthe magnetic fields because the fields bend around between adjacentmagnetic dipole moments.

Assuming a spherical shape, a magnetic particle place within a magneticfield distribution can be described to experience a magnetic force{right arrow over (F)}_(B) according to Eq. (1) presented below:

$\begin{matrix}{{\overset{\rightarrow}{F}}_{B} = {\frac{V\; \chi}{\mu_{0}}\left( {\overset{\rightarrow}{B} \cdot \overset{\rightarrow}{\nabla}} \right){\overset{\rightarrow}{B}.}}} & (1)\end{matrix}$

where V is the volume of the particle (m³), χ is the magneticsusceptibility (unit-less) and μ₀ is the vacuum permeability (V×s/A×m).{right arrow over (B)} is the magnetic field at the location of theparticle. Strong magnetic fields and large gradients of magnetic fieldsprovided by the array of magnets 102 and 103 can apply a large force of{right arrow over (F)}_(B) to the magnetic particle than the case ofsandwiching magnets without alternating dipoles or a magnet with acheckerboard pattern of magnetic dipoles positioned on only one side ofthe channel 506.

Accordingly, referring back to FIG. 4, the arrangement of magneticdipole moments 231, 232, 252, and 252 can provide a strong magneticforce for isolating particles. For example, when sample fluid flows indirection 412 of channel 506, magnetic particles or particles 420 withattached magnetic beads 422 can experience a strong magnetic force{right arrow over (F)}_(B) and become attracted towards either the arrayof magnets 102 or 104. On the other hand, particle 430, is not magneticand does not experience the magnetic force {right arrow over (F)}_(B),and therefore flows through channel 560 without becoming attached towalls 440 of the microfluidic channel layer 204. For example, theparticles 420 can be abundant blood cells and the particles 430 can betarget tumor cells. In the above approach, the blood cells can beeffectively depleted while the target tumor cells pass through thechannel 506.

The strong magnetic fields and large gradients of the magnetic fields ofthe magnets 231, 232, 251 and 252 can lead to a higher probability ofparticles 420 becoming attached to walls 440. Moreover, the relativelystronger magnetic fields can allow the fields to extend a relativelylonger distance. Thus, the magnets 231, 232, 251 and 252 can efficientlyattract particles 420 with attached magnetic beads 422 at a relativelylonger distance. The long distance allows the presence of walls 440(e.g., floor and ceiling of channel 506), plate 202 (e.g., glass plate)and plate 450 (e.g., glass plate) between the magnets 231, 232, 251 and252 and the channel 506, respectively, and thereby avoiding directcontact of the sample fluid with the magnets 231, 232, 251 and 252.Accordingly, particles 420 do not get directly attached to the magnets231, 232, 251 and 252, and the particles 420 can later be easily removedfrom the walls 440 by removing the magnets 231, 232, 251 and 252. Inthis approach, the particles 420 can be easily isolated and collected,while the magnets can be easily reused for other sample fluids without acleansing process. In addition, the presence of walls 440, plate 202 andplate 450 can reinforce the structural rigidity of the microfluidicdevice 110.

In come embodiments, the channel 506 can have a height 451 of 50 μm, or0.1 mm or more (e.g., 0.2 mm or more, 0.5 mm or more, 1 mm or more 1.5mm or more) due to the long range of magnetic fields provided by themagnets 231, 232, 251 and 252. A larger height 451 can lead to a higherthroughput of the sample fluid. Therefore, by increasing the height 451,which is possible due to the strong magnetic force, the system 100 canprovide a high throughput isolation of particles. The height 451 canalso be selected depending on the diameter (e.g., 40 μm) of the largestparticle in the sample fluid. For example, the height 451 can be atleast 20% larger than the largest diameter. In some cases, the channel506 can have a height between 50 μm and 5 mm (e.g., between 50 μm and 1mm, between 1 mm and 2 mm, between 2 mm and 3 mm, between 3 mm and 4 mm,or between 4 mm and 5 mm). Correspondingly, the distance between thearrays of magnets 102 and 104 can be between 50 μm and 5 mm.

The long range of magnetic fields can attract particles 420 even withthe presence of the thicknesses of plates 202 and 450 and walls 440. Incertain embodiments, the thicknesses of plates 202 and 450 can be 300 μmor more (e.g., 500 μm or more, 750 μm or more, 1 mm or more, 1.5 mm ormore). The thickness of walls 440 can be 100 μm or more (e.g., 300 μm ormore, 500 μm or more, 750 μm or more, 1.0 mm or more).

FIG. 5A is a schematic diagram showing the microfluidic device 110according to coordinate 292. The microfluidic device 110 can beconfigured to receive sample fluid through its inlet 502. The samplefluid can flow from the inlet 502 towards outlet 504. During the flow,the sample fluid passes one or more channels 506 and then through aparticle capture zone 508. The arrays of magnets 102 and 104 sandwich aregion 510 of the one or more channels 506. Thus, the magnetic fieldsprovided by the arrays of magnets 102 and 104 can isolate magneticparticles or particles with attached magnetic beads as the sample fluidpasses through the one or more channels 506. Remaining particles thatare not attracted by the magnets or particles with attached beads thatdo not get attached to walls by chance flow into the particle capturezone 508. In this example, a channel 506 has one or more windings toextend the length of the channel 506 to capture as many particles withattached beads as possible. This can lead to a high enrichment ofparticles 430.

Particles that pass through the particle capture zone 508 reach outlet504 by way of a fluid manifold 505, which is positioned between theparticle capture zone 508 and the outlet 504. In this example, the fluidmanifold 505 is a passage between the particle capture zone 508 and theoutlet 504.

FIG. 5B is a schematic diagram showing another example of a microfluidicdevice 510. In this example, a channel 506 is a straight channel withoutany winding. Such a configuration can be easier to fabricate than thedesign shown in FIG. 5A. Although, the channel 506 of the microfluidicdevice 510 has a relatively shorter length than the case shown in FIG.5A, the isolation of particles with attached magnetic beads can besufficiently effective due to the strong magnetic force provided byarrays of magnets 102 and 104 sandwiching region 510. Accordingly, thedesign and arrangement of the arrays of magnets 102 and 104 can lead toa simpler design of a microfluidic device with easier and reduced costsof manufacturing.

In some embodiments, herringbone mixers (also referred as “chaoticmixers”) can be structured on the floor or ceiling within channel 506 togenerate turbulence in the flow of the sample fluid. The turbulence canincrease the chance of particles being located closer to the top orbottom walls of the channel(s), and thereby becoming attracted to themagnets. However, when the arrays of magnets 102 and 104 as describedherein are positioned above and below the microfluidic device, thenumber of herringbone mixers can be reduced or eliminated due to thepresence of strong magnetic forces provided by the magnets. Thisapproach can be advantageous by simplifying the fabrication processand/or cost due the reduction or absence of the herringbone mixers.

FIG. 6A is a schematic diagram showing a portion of the microfluidicdevice 110 described in FIG. 5A. Image 600 is a zoomed in view of theparticle capture zone 508, which includes a plurality of particlecapture sites 601. As the sample fluid flows towards the outlet 504,remaining particles 430 can be captured in the plurality of particlecapture sites 601. In some embodiments, each particle capture site 601can include a receptacle sized to receive and confine one or more of theremaining particles 430. For example, in some embodiments, a singleparticle capture site 601 can have a receptacle having a size similar toa size of a target among the remaining particle 430. For instance, thereceptacle can have a width, height, or cross-section approximatelyequal to a width, height, or cross-section, respectively, of one of theremaining particles. As an example, the receptacle can have a width thatis between about 10 μm to 50 μm. In some cases, the size of thereceptacle can be larger than an average diameter of the remainingparticles 430 (e.g., having a size 10% larger, 20% larger, 30% larger,40% larger, 50% larger, or more than 50% larger than the size of thetarget among the remaining particles 430). In some cases, thecross-section of the receptacle can be larger than an averagecross-section of the remaining particles 430 (e.g., a cross sectionalarea 10% larger, 20% larger, 30% larger, 40% larger, 50% larger, or morethan 50% larger than the cross-sectional area of the target among theremaining particles 430). In some cases, the shape of the receptacle canbe similar to the shape of the expected shape of the remaining particles430. In example, in some cases, the shape of the receptacle can begenerally circular, ovular, or elliptical, depending on the shape of theremaining particles 430. In some implementations, the receptacles of oneor more of the particle capture sites can each be sized to receive andcontain a single one of the remaining particles 430. This arrangementallows individual remaining particles 430 to travel into a particlecapture site 601, and become trapped within the particle capture site601. The trapping can be due to the physical size and shape of theparticle capture site 601.

FIG. 6B shows a top view the particle capture zone 508. In this example,the sample fluid flows in direction 680. In some embodiments, theparticle capture zone 508 includes a plurality of particle capture sites601, 602, and 603 of different sizes. For example, the particle capturesite 601 has two walls with an opening 641 in between. The separation682 between the two walls of site 601 defines a receptacle, which can becan be about 5% to 15% larger (e.g., 10% to 20% larger, 15% to 25%larger, 20% to 30% larger, 30% to 40% larger) than the diameter oftarget particle 631. The separation 682 can allow the particle capturesite 601 to capture a single particle 631.

On the other hand, a width of opening 641 can be about 10% to 20%smaller (e.g., 20% to 30% smaller, 30% to 40% smaller) than the diameterof target particle 631. The opening 641 can allow particles smaller thanthe particle 631 to pass through the particle capture site 601 withoutbeing captured. For example, particles 632 and 633, being smaller thanparticle 631, passes particle capture sites 601. The particle 632 can becaptured by particle capture site 602, which has an opening 642 forallowing the passage of particles smaller than particle 632. Theparticle 633 can be captured by particle capture site 603, which has anopening 643 for allowing passage of particles smaller than the particle633. The openings 641-643 can also mitigate clogging of particle capturesites 601-603. Generally, the size of each particle capture site can beselected so that the site can dominantly capture a single particle. Inthis approach, the particle capture sites 601-602 can function as asize-based sorter. In some cases, the separation 682 and the opening 641can be aligned parallel or substantially parallel to a direction offluid flow in the particle capture zone 508 (e.g., within 5°, within 3°,within 1°), such that the receptacle and the opening 641 of eachparticle capture site are also aligned parallel or substantiallyparallel to a direction of fluid flow in the particle capture zone 508.

In some embodiments, a plurality of particle capture sites withidentical dimensions can be arranged in one or more rows. For example,the particle capture sites 601 can be arranged in multiple rows of 30 ormore (e.g., 10, 25, 50, 75, 100, 250, 500 or more, e.g., 1000 or more).Each row can include particle capture sites 601 of 25, 50, 75, 100, 150,250 or more (e.g., 500 or more, 1000 or more).

Generally, a particle capture site can capture a particle due to thephysical shape of the particle capture site rather than relying onbinding moieties. This approach can be advantageous, because thecaptured particle can be easily manipulated as will be described infurther detail below. On the other hand, a particle captured utilizingbinding moieties, e.g., antibodies, can be difficult to detach from acapture site for subsequent manipulation and characterization.Accordingly, in some embodiments, instead of binding moieties, particlecapture sites can be coated with surfactants such as Pluronic® F-127,Tween® 80, Tween® 20, bovine serum albumin (BSA), and other cell culturelevel surfactant to reduce sticking of particles onto the particlecapture sites. In certain embodiments, the particle capture sites can bemade from glass, plastic, acrylic glass, poly-methyl methacrylate(PMMA), or other materials that reduce non-specific binding of particlesto the surfaces of the particle capture sites as well as the walls(including floor and ceiling walls) of the entire microfluidic device.

FIG. 6C is a schematic diagram of another example of a particle capturezone 670, which includes a plurality of particle capture sites 604. Assample fluid flows in direction 680, particle capture sites 604 cancapture particle 610 while allowing smaller particle 612 to passthrough. View 620 shows a zoomed image of a portion of the particlecapture zone 670. As illustrated, each particle capture site 604 isdefined by a recess or notch 620 which has a size configured to capturea single particle 610. The particle capture site 604 also has an openingwith width 622 and height 624 that allows particle 612 to pass through.The size of notch 620, width 622 and height 624 can be selected based onthe diameters of particles 610 and 612 to be captured and passedthrough.

As mentioned earlier, system 100 can include the tweezer device 120,which can be used for manipulation of particles in the sample fluid.Referring back to FIG. 6B, side-view 660 depicts a zoomed side-view(instead of a top view) of particle capture size 601 with its capturedparticle 631. In this example, tweezer device 120 is an optical tweezerutilizing a focused optical beam 662 to trap the particle 631 within thebeam waist of the optical beam. The optical beam 662 is focused by amicroscope objective (not shown). Electric field gradients of theoptical beam attract the particle 631 within the beam waist. Theparticle 631 can be lifted up and moved around by moving the position ofthe focused beam waist in 3-dimensional space. The focused beam can bemoved by displacing the microscopic objective or adjusting the beamdirection using actuators. The displacement or adjustment can be carriedout manually by a user or through a computer controlled motor asdescribed below.

Optical tweezers for manipulating materials that are useful in thepresent methods and systems are described, for example, in U.S. Pat.Nos. 7,104,659, and 7,759,635. Acoustic tweezers for manipulatingmaterials that are useful in the present methods and systems aredescribed, for example, in Ding, Xiaoyun, et al., “On-chip manipulationof single microparticles, cells, and organisms using surface acousticwaves.” Proceedings of the National Academy of Sciences 109.28 (2012):11105-11109, and in Li, Ying, et al. “A simple method for evaluating thetrapping performance of acoustic tweezers.” Applied physics letters102.8 (2013): 084102. The contents of each of the foregoing patents andpublications are incorporated herein by reference in their entirety.

FIG. 7A is a schematic diagram of an example of a tweezer device 710that can be used in system 100. The tweezer device 710 can include anoptical source (e.g., laser source) to provide an optical beam. A mirror704 can reflect the optical beam towards a lens device 706, whichfocuses the optical beam into one or more particle capture sites (notshown) in particle capture zone 508. Other optical components that canbe used for beam shaping and steering are not shown, but are well knownto those of skill in this field. In some embodiments, the lens device706 can be a single microscopic objective used to focus the optical beamfor optical trapping. In certain embodiments, the lens device 706 can bea microlens array which splits the optical beam into a plurality offocused beams. Each of the focused beams can be used for opticaltrapping of particles captured in the particle capture zone 508. In thisapproach, the tweezer device 710 can provide a plurality of opticaltraps.

The mirror 704 can be a planar mirror for directing the optical beamtowards the lens device 706. In some embodiments, the mirror 704 can bea spatial light modulator (SLM), which can direct different portions ofthe optical beam independently. For example, the mirror 704 can be anarray of MEMS mirrors, where each mirror can steer different portions ofthe optical beam through the lens device 706 in order to manipulatedifferent optical traps independent of one another.

The tweezer device 701 is often used in conjunction with an imagingdevice 708 that can be used to image the particle capture zone 508. Forexample, the imaging device 708 can include a 2D charge-coupled device(CCD) camera that can monitor individual particle captures sites andcaptured particles in the particle capture zone 508. In someembodiments, the imaging device 708 can send image data to a controller710.

In certain embodiments, the controller 710 can process and visualize theimage data on a screen. A user may then monitor the screen and inputinformation using an input device (e.g., keyboards, mouse, andtouchscreen) so that the controller 710 sends out control signals to themirror 704 and/or other optical components in the tweezer device 710.For example, the user can visually monitor a touchscreen and select atarget particle through the touch screen for analysis. In certainembodiments, the control signals can activate actuators, for example,for controlling MEMS mirrors in mirror 704 or other optical componentsto steer a focused optical beam to trap the selected target particle formanipulation.

In some embodiments, the controller 710 can process the image data andselect a target particle to trap and manipulate without an input fromthe user. For example, the controller 710 can determine which targetparticles to select based on the size of the corresponding particle siteand/or based on a reporter group or marker, e.g., color, fluorescence,or radioactivity, on or of the particles. For example, a color can bedue to staining of a specific type of particles. The controller 710 cancompare the size and color information to library information stored inthe controller 710 to determine the selection and manipulation as isknown in the relevant field.

FIG. 7B is a schematic diagram of another example of a tweezer device720, which can be used in system 100. The tweezer device 720 includestwo optical sources 702 which two beams can be used to interfere andform an optical lattice. A focused beam point in the optical lattice canbe used to capture and manipulate a particle in particle capture zone508. In some embodiments, mirrors 704 can include SLMs that canmanipulate individual lattice points in the optical lattice forcontrolling individual or a group of trapped particles. In certainembodiments, the tweezer device 720 can use computer-generated hologramsto create a pattern of the optical lattices to capture selectedparticles.

Referring to FIG. 8, a schematic diagram shows the top view of theparticle capture zone 508 described in relation to FIG. 6B. In someembodiments, tweezer device 120 can be configured to trap a singleparticle (e.g., particle 631) at a given time, lift up and release theparticle for subsequent analysis. In certain embodiments, the tweezerdevice 120 can be configured to trap a plurality of particles at thesame time. For example, particles in regions 804, which have differentsizes, can be trapped, lifted up and displaced at the same time. Asanother example, particles in regions 802, which have the same size, canbe trapped, lifted up and displaced at the same time. In some example,the tweezer device 120 can select particles with same stain color forsubsequent analysis.

FIGS. 9A-9C are schematic diagrams of images showing a process formanipulating a target particle. In FIG. 9A, target particle 902 inparticle capture site 601 is stained in a green color. The staining canbe achieved before the sample fluid is introduced into microfluidicdevice 110. In some embodiments, the staining can be achieved after thesample fluid is in microfluidic device 110 and the target particle 902is captured in particle capture site 601. For example, the stainingprocess can include attaching targeting particles 902 (e.g.,intracellular, extracellular markers) with fluorescent antibodies,drugs, or chromophores. In FIG. 9B, optical beam 662 is focused tooptically trap the target particle 902. By moving the beam waist of theoptical beam 662 upwards, the trapped target particle 902 can be lifted,while other particles 430 remain captured.

In FIG. 9C, the optical beam 662 is removed so that target particle 902is released from trapping. When flushing fluid flows in direction 680,the released target particle 902 can follow the follow and becometransported to outlet 504. At this step the flush fluid can be the sameor different from the sample fluid depending on the analysis method. Forexample, in some applications, it can be desirable to use a flush fluidwithout any particles to improve purity of the collected samples. Incertain embodiments, the flush fluid can be same sample fluid withoutchanging to another fluid. The absence of changing fluid can allow fastcollection of samples. In some embodiments, the flush fluid can be usedfor high throughput application. In certain embodiments, the opticalbeam 662 can move the target particle 902 with high precision to aspecific site for analysis.

FIG. 10 is a schematic diagram showing a portion of system 100.Particles can reach outlet 504 by flowing within sample fluid. In someembodiments, particles selected and released by tweezer device 120 canreach the outlet 504 by the flushing or washing fluid. These particlescan be transported to receiver device 114 through coupling 115. Incertain embodiments, the receiver device 114 can include a plurality ofcontainers 1002-1008. Each container can be used to receive and containa single particle, a group of particles, same type of particles ordifferent type of particles for characterization. For example, tweezerdevice 120 can trap and release a single target particle which istransported to the receiver device 114. After the container receives thetransported single target particle, a user or a motorized staged canadjust the containers so that container 1004 can receive the nextsamples. For example, next the tweezer device 120 can trap and releasetarget particles which are transported to the receiver device 114. Afterreceiving a plurality of target particles, the container 1004 can beremoved for characterization of the contained target particles.

Fabrication

Microfluidic device 110, and other similar devices as described herein,can be manufactured using a variety of fabrication processes, e.g.,processes known to those of skill in the relevant fields. For example,in some embodiments, an injection molding process is used to fabricatethe microfluidic device 110. For example, material such as glass, PDMS,or melted plastic can be injected into a predetermined mold defininginlet 502, outlet 504, channels 506, particle capture zone 508, and itsparticle capture sites. After cooling and hardening of the injectedmaterial, further process such as drilling and/or cutting can beimplemented to refine the pattern formed in the microfluidic device 110.The patterned layer is then bonded to another flat substrate (e.g.,glass, plastic, PDMS) to form a complete, sealed microfluidic device110.

In some embodiments, etching or photolithography processes can beimplemented to fabricate the microfluidic device 110. For example, afilm of PDMS can be spin coated on a substrate, and then light is usedto transfer a pattern from a photomask to the PDMS film. The pattern candefine the various openings (e.g., inlet 502, outlet 504, channels 506,particle capture zone 508 and particle capture sites) of themicrofluidic device 110.

In certain embodiments, imprint lithography can be implemented tofabricate the microfluidic device 110. For example, a mold definingvarious openings (e.g., inlet 502, outlet 504, channels 506, particlecapture zone 508 and particle capture sites) can be pressed against animprint resist while being cured by heat or ultra-violet (UV) light.

In some implementations, during operation of the system 100, themicrofluidic device 110 can be mounted on a device holder. The holdercan include two slabs (e.g., a top and bottom slab), with each slabcontaining the magnet arrays 102 and 104. The microfluidic device 110can be positioned and held in place between the slabs through amechanical locking mechanism (e.g., through screws, notches, pegs, pins,or clamps), the magnetic pulling between the magnet arrays, or both. Todefine the fluidic ports, pairs of cylindrical magnets (e.g., magnets210 and 212; and/or magnets 220 and 222) can be aligned at the openings502 and 504 of the microfluidic device 110. The fluidic ports can besecured to the microfluidic device through the magnetic pulling betweenthe pair of magnets 210 and 212, and/or the pair of magnets 220 and 222.

By separating the microfluidic device and the magnetic system, thismodular scheme can simplify the device fabrication and facilitate thesystem setup. It can also reduce the operation cost, as the deviceholder, magnet arrays and the magnetic ports can be repeatedly used.

General Methodology

Referring to FIG. 11, a flow chart 1100 depicts examples of operationsteps for isolating particles using a system described herein, e.g.,system 100. Operations include providing a first and a second array ofmagnets 102 and 104, where the first and second arrays of magnets 102and 104 are positioned to sandwich a region including a channel 506 of amicrofluidic device 1110 (1110). In some embodiments, the first andsecond arrays of magnets 102 and 104 include magnetic dipole momentsarranged in alternating order, such that adjacent magnets have dipolemoments aligned in opposite directions. The magnets of the first andsecond array can include magnets made of materials such as NdFeB, SmCo,FePt, MnFe₂O₄, CoFe₂O₄, NiFe₂O₄, ZnMnFe₂O₄, or iron oxide.

Operations also include providing a sample fluid including particlesinto an inlet 502 of the microfluidic device 110 (1120). In someembodiments, the particles can include tumor cells, blood cells such asleukocytes and biomarkers. Some of these particles can be bound bymagnetic beads and/or fluorescent beads. In certain embodiments, some ofthe particles can be stained. For example, cancer cells can bepre-stained with 1:2000 Hoescht 33258 (Invitrogen).

A first particle bound to a magnetic bead can be separated fromremaining particles in the sample fluid (1130). This can be achieved byflowing the sample fluid through the channel 506, where the firstparticle is separated by magnetic fields provided in the region betweenthe first and second arrays of magnets 102 and 104. Strong magneticfield strength and large gradients of magnetic fields can provide a longrange force to attract particles bounded with magnetic beads.

Operations also include capturing a second particle not bound to amagnetic bead from the remaining particles in the sample fluid into aparticle capture site of the microfluidic device 110 (1140). In someembodiments, the microfluidic device can include a plurality of particlecapture sites of 100 or more (e.g., 500 or more, 1000 or more, 10000 ormore). At least some of the particle capture sites can have a size andshape designed to capture a single particle. Different particle capturesites can have different sized designed to capture particles withdifferent sizes.

The captured second particle can be displaced using an optical tweezerdevice (1150). The optical tweezer device can be configured to trap asingle particle. In certain embodiments, the optical tweezer device canprovide multiple optical traps configured to trap multiple particles atthe same time. The optical tweezer device can displace the secondparticle out of its particle capture site. Then the displaced secondparticle can be transported to an outlet 504 by releasing it from theoptical tweezer device.

Operations also include collecting the displaced second particle in areceiver device 114 (1160). The released second particle in operation(1130) can be transported from the outlet 504 to a receiver device 114.In some embodiments, the receiver device 114 can include a plurality ofcontainers, each receiving different particles released by the tweezerdevice 120. In this approach, a user can collect target particles inseparate containers for subsequent characterization.

Hardware and Software Implementation

FIG. 12 is a schematic diagram showing an example of a controller 710,which may be used with the techniques and systems described herein. Asmentioned above, the controller 710 can be used to receive image dataand control beam directions of a tweezer device. The controller 710 caninclude a processor 1202, memory 1204, a storage device 1206, andinterfaces 1208 for interconnection. The processor 1202 can processinstructions for execution within the controller 710, includinginstructions stored in the memory 1204 or on the storage device 1206.For example, the instructions can instruct the processor 1202 toidentify characteristics of captured particles based on, e.g., size andcolor. The processor 1202 can be configured to send out control signalsto adjust beam directions of the tweezer device.

The memory 1204 can store information of parameters of the tweezerdevice. For example, the information can relate actuator positions tofocus positions of an optical beam. The storage device 1206 can be acomputer-readable medium (e.g., a hard disk, an optical disk, a flashmemory or solid state memory device), which can store information ofcharacteristics (e.g., color, size, shape) of predetermined particles.The process 1202 can compare the stored information to image data toidentify the types of captured particles. The storage device 2605 canstore instructions that can be executed by processor 1202 describedabove. In certain embodiments, the storage device 2605 can storeinformation described in relation to memory 1204.

In some embodiments, controller 710 can include a graphics processingunit to display graphical information (e.g., using a graphical userinterface (GUI) or text interface) on an external input/output device,such as display 1216. The graphical information can be displayed by adisplay device (e.g., a CRT (cathode ray tube) or LCD (liquid crystaldisplay) monitor) for displaying information. A user can use inputdevices (e.g., keyboard, pointing device, touch screen, speechrecognition device) to provide input to the controller 710. For example,the input can identify which captured particle to manipulate. Based onthe input, the controller 710 can control the system as described above.

Various embodiments of the systems and techniques described herein canbe implemented as one or more computer programs that are executableand/or interpretable on the controller 710. These computer programs(also known as programs, software, software applications or code)include machine instructions for a programmable processor, and can beimplemented in a high-level procedural and/or object-orientedprogramming language, and/or in assembly/machine language. For example,computer programs can contain the instructions that can be stored inmemory 1204 and storage 1206 and executed by processor 1202 as describedabove. As used herein, the terms “computer-readable medium” refers toany computer program product, apparatus and/or device (e.g., magneticdiscs, optical disks, memory, Programmable Logic Devices (PLDs)) used toprovide machine instructions and/or data to a programmable processor,including a machine-readable medium that receives machine instructions.

In some implementations, the controller 710 can be interconnected withother components. For example, the controller 710 can be communicativelycoupled to one or more server computers 1220, one or more laptopcomputers 1222, and/or one or more desktop computers 1224. As anotherexample, the controller 710 can be communicatively coupled to one ormore peripherals (e.g., input devices 1226, printers 1228, or scanners1230). In some cases, the controller 710 can be communicatively coupledto one or more computer systems 1234 through a network (e.g., through anetwork switch 1232). The controller 710 can transmit data to, andreceived from, each of these interconnected components in order toperform some or all of the functions described above.

Generally, controller 710 can be implemented in a computing system toimplement the operations described above. For example, the computingsystem can include a back end component (e.g., as a data server), or amiddleware component (e.g., an application server), and/or a front endcomponent (e.g., a client computer having a graphical user-interface),or any combination therefor, to allow a user to utilized the operationsof the controller 710.

General Applications

Generally, system 100 can be used as a platform for isolating particles,such as rare target cells (e.g., circulating tumor cells, circulatingstem cells, or fetal cells circulating in maternal blood) directly fromthe whole blood samples. The strong magnetic forces provided by thearrays of magnets sandwiching a microfluidic channel as described hereincan be used to immuno-magnetically deplete abundant host cells such asleukocytes while enabling target cells such as cancer cells to passthrough the magnetic fields. These target cells can be individuallycaptured by physical barriers, and subsequently analyzed in situ forcomprehensive and multifaceted evaluation, including single cellenumeration and imaging, molecular and genetic profiling, anddrug-treatment responses. Moreover, a tweezer device can be used to liftand retrieve captured particles (e.g., cancer cells) which can befurther transported for downstream off-chip analyses.

As an example, a biological sample (e.g., a blood sample, a cerebralspinal fluid sample, or any other biological sample) can include severaldifferent types of cells and other particles, only some of which may beof interest for a particular study. The system 100 can be used toimmuno-magnetically deplete this biological sample, such that thatunwanted cells are separated from the biological sample, while cells ofinterest remain in the biological sample and become immobilized withinthe capture zone of the system 100. Once captured, these cells can beindividually cultured and/or examined in order to further investigatethe properties of each cell.

In some cases, cells that have been immobilized within the capture zonecan be examined through immunostaining. As an example, captured cellscan be exposed to fluorescently labeled particles (also calledfluorescent markers) that exhibit binding specificity to particularbiomarkers of interest. These particles can include, for example,molecules having targeting moieties specific to certain biomarkers(e.g., antibodies, enzymes, cellular receptors, or other targetingmolecules), in which the molecules are bound to fluorophores (e.g.,phycoerythrin (PE) or fluorescein isothiocyanate (FITC)). Captured cellscan be exposed to these fluorescently labeled particles, for instance,by flowing the fluorescently labeled particles across the capture zoneof the system 100, such that the particles are allowed to bind tocaptured cells that express these biomarkers. The capture zone of thesystem 100 can then be examined to determine the presence of thebiomarkers of interest (e.g., by optically exciting the fluorophores,capturing images of the resulting fluorescence, and observing theintensity of the resulting fluorescence). As individual cells can becaptured in each of the capture sites of the capture zone, these cellscan be examined for the presence or absence of the biomarker of interestwith single cell resolution Thus, the system 100 can be used to receivea biological sample, increase the relative abundance of cells ofinterest within the biological sample through immuno-magneticseparation, and identify individual cells expressing a particularbiomarker of interest. Individual cells (e.g., cells expressing thebiomarker of interest) can then be collected for further study.

In some cases, implementations of this system can be used to investigatethe relative population of certain types of cells within a biologicalsample. As an example, a biological sample may include several differenttypes of cells. One or more of these cell types may beimmuno-magnetically depleted, such that they are removed from thebiological sample. The remaining cells types can be captured within thecapture zone of the microfluidic device, then analyzed usingimmunostaining to ascertain if the captured cells express one or moreparticular biomarkers. The presence or absence of these biomarkers canbe used to deduce the type of cells that have been captured within thecapture zone. As the capture zone contains several different capturesites, each immobilizing a different individual cell, an immunostaininganalysis of the capture sites of the capture zone can reveal informationregarding the relative abundance of each type of cell within thebiological sample. This can be useful, for example, for lymphomadetection, where several different sub-types of lymphoma each express aparticular biomarker or combination of biomarkers. Implementations ofthis system can be used to differentiate between each of these differentsub-types. Although lymphoma is provided as an example, this is merelyillustrative. In practice, implementations of the system 100 can be usedto differentiate between other types of disease cell sub-types, or anyother type of cell.

In some applications, implementations of the system 100 can be used fordrug screening. As an example, cells of interest that have been capturedwithin the capture zone of the system 100 can be incubated, then exposedto one or more different drugs and/or incubated with drugs for varyingincubation times. In some cases, these drugs can be introduced into thecapture zone by flowing the drugs through the microfluidic device (e.g.,by introducing the drug into an inlet of the microfluidic device, andallowing the drug to flow throughout the microfluidic device and out ofthe outlet of the microfluidic device. In some cases, one or more ofthese drugs can be introduced into the capture zone by additional inletsof the microfluidic device that are in direct fluid communication withthe capture zone of the microfluidic device. The captured cells can beindividually exampled before and/or after incubation with the drugs(e.g., using immunostaining) in order to ascertain the effect of thosedrugs on each captured cell. In some cases, different regions of thecapture zone can be exposed to different drugs and/or differentconcentrations of drugs, such that a wide array of therapeuticconditions can be investigated simultaneously within a single capturezone.

For example, in some cases, certain regions of the capture zone can beexposed to a first concentration of a drug, while another region of thecapture zone can be exposed to a second concentration of a drug.Accordingly, captured cells within each of the regions will be exposedto different concentrations of the drug. The captured cells can beindividually examined before and/or after incubation with the drugs(e.g., using immunostaining) in order to ascertain the effect of varyingconcentrations of the drug on the captured cells.

As another example, in some cases, certain regions of the capture zonecan be exposed to a first drug, while another region of the capture zonecan be exposed to a second drug. Accordingly, captured cells within eachof the regions will be exposed to different drugs. The captured cellscan be individually exampled before and/or after incubation with thedrugs (e.g., using immunostaining) to ascertain the effect of differentdrugs on the captured cells.

In some cases, the capture zone or region can be exposed to a gradientof different drugs and/or different drug combinations. In some cases,this can be achieved by flowing two or more drugs and/or drugconcentrations in parallel across the capture zone (e.g., by introducingthe drugs into the inlet 502 and/or into one or more additional inletsdistributed across an end of the capture zone in a direction orthogonalto a direction of fluid flow through the capture zone). As the drugsflow across the capture zone, the drugs mix and create a gradient withinthe capture zone. The captured cells can be individually exampled beforeand/or after incubation with the drugs (e.g., using immunostaining) toascertain the effect of different drugs and/or drug concentrations onthe captured cells. In some cases, as the drugs flow across the capturezone, the drugs do not mix (or do not substantially mix) due to laminarflow. In these cases, the drugs do not create a continuous gradient.However, this combination of different drug (or drug concentration)still creates a gradient (e.g., a gradient with a discrete number ofdifferent drugs or drug concentrations) within the capture zone.

As an example, as shown in FIG. 25A a cell 2502 that has been capturedwithin a capture site 2504. Once captured, the cell 2502 can beincubated within the capture site 2504. As shown in FIG. 25B, a capturezone 2506 can include an array of spatially distributed capture sites2504. Different regimens of drugs (e.g., different concentrations ofdrugs) can be flowed across the capture sites 2504, such that each ofthe capture sites 2504 is exposed to a spatially-dependent drug regimen.As shown in FIG. 25C, the capture sites 2504 can each be analyzed (e.g.,using immunostaining), in order to investigate the effect of eachtreatment regimen on the capture cells.

Using the system 100 for drug screen can be beneficial, for example, forassessing the effectiveness of treating various types of lymphoma.Implementations of this system can be used to treat various types oflymphoma cells with one or more therapeutic drugs, then assess theeffectiveness of those drugs in binding to each of the different celltypes and effecting a response. Although lymphoma is provided as anexample, this is merely illustrative. In practice, implementations ofthe system 100 can be used to assess the effectiveness of treatingvarious other types of diseased cells.

In some applications, implementations of the system 100 can be used toscreen cells through particle-cycling (e.g., antibody-cycling,enzyme-cycling, or cellular receptor-cycling). As described above, insome cases, cells that have been immobilized within the capture zone canbe examined through immunostaining. For example, fluorescent labeledparticles having targeting moieties specific to a first type ofbiomarker can be flowed across the capture zone of the system 100, suchthat the fluorescent particles are allowed to bind to captured cellsexpressing the first type of biomarker. The capture zone of the system100 can then be examined in order to determine the presence of the firsttype of biomarker. After examination, the fluorescent particles bound tothe captured cells can be cleaved from the captured cells (e.g., usingan elution buffer or photo-cleaving), and removed from the capture zone.As a result, the captured cells remain immobilized within the capturesites, but are no longer bound to any fluorescently labeled particles.Subsequently, a second group of fluorescently labeled particles havingtargeting moieties specific to a different type of biomarker can beflowed across the capture zone of the system 100, such that the secondgroup of fluorescently labeled particles are allowed to bind to capturedcells expressing the second type of biomarker. The capture zone of thesystem 100 then can be examined in order to determine the presence ofthe second type of biomarker. In this manner, the same captured cellscan be investigated using multiple “cycles” of fluorescently labeledparticles, where each cycle reveals the presence or absence of adifferent biological marker. In some cases, each cycle can includemultiple different types of fluorescently labeled particles, eachspecific to a different particular biomarker, and each fluorescing at adifferent particular wavelength. In this manner, the presence or absenceof several different biomarkers can be examined simultaneously. Afterexamination, these particles can be cleaved from the captured cells, andone or more additional fluorescently labeled particles can be flowedacross the captured cells. This cycle can be repeated any many times asdesired (e.g., one, two, three, four, or more times) to examine thepresence or absence of any number of different biological markers.

As an example, as shown in FIG. 26A a cell 2602 that has been capturedwithin a capture site 2604. As shown in FIG. 26B, once the cell 2602 hasbeen captured, a first fluid sample containing a first type offluorescently labeled particle 2606 is flowed across the capture site2604. The fluorescently labeled particles 2606 are specific to aparticular biomarker expressed by the cell 2602. Thus, as shown in FIG.26C, one or more of these fluorescently labeled particles 2606 adhere tothe cell 2602. While the fluorescently labeled particles 2606 areadhered to the cell 2602, the capture sites 2604 can each be analyzed(e.g., using immunostaining), to investigate the presence or absence ofthe fluorescent marker, and consequently, the presence or absence of thebiomarker on the cell 2602. As shown in FIG. 26D, an elutant is flowedacross the capture site 2604. This elutant cleaves the fluorescentlylabeled particles 2606 from the cell 2602, removing the from the cell2602 and from the capture site 2604. Subsequently, a second fluid samplecontaining a second type of fluorescently labeled particle is flowedacross the capture site 2604, where the second type of fluorescentlylabeled particles are specific to a different particular biomarkerexpressed by the cell 2602. Thus, this staining and cleaving process canbe “cycled” to examine the presence or absence of any number ofdifferent biological markers.

Although the examples of applications described herein use fluorescentlylabeled particles, some implementations can also include the use ofother types of particles or labels, either in addition to or instead offluorescently labeled particles. For example in some cases capturedcells can be stained with particles labeled with non-fluorescing dyes(e.g., dyes that exhibit a particular color in the visible spectrum) ordirectly with the dyes themselves without the use of additionalparticles. The use of combinations of fluorescently labeled particlesand non-fluorescing particles is also possible, depending on theimplementation.

The disclosed systems and techniques can be used for rare cellenrichment from native biological samples (e.g., whole blood, urine,spinal fluid). System 100 can be used as a sample preprocessor formolecular analysis systems, for example, used for gene sequencing. Insome applications, the techniques can be used to characterize ascitestumor cells (ATCs) and monitoring of treatment of ovarian cancer withoutinvasive surgical biopsies.

EXAMPLES

The methods and systems described herein are further illustrated usingthe following examples, which do not limit the scope of the claims.

Example 1—Arrays of Magnets

FIGS. 13A and 13B are schematic diagrams showing two configurations of5×5 array of magnets and their calculated magnetic field distribution.The magnets were modeled to be made from NdFeB material with asaturation magnetization M=750 kA/m. Referring to FIG. 13A, 5×5 arrays1329 and 1349 are arranged according to coordinate 291. Each magnet ofthe arrays 1349 and 1349 has a length 1311 of 1 mm. Hence, magneticdipole moments alternate with a period of 2 mm in the y-direction. Theclosest surfaces between the arrays 1329 and 1349 have a separationdistance 1312 of 1.4 mm. Image 1302 shows the calculated absolute valueof magnetic field component in the z-direction (|Bz|) for region 1301between the arrays 1329 and 1349. z=0 mm corresponds to the bottomsurface of array 1329 and z=1.4 mm correspond to the top surface ofarray 1349. Regions 1304 have a large absolute value of magnetic fieldcomponent in the z-direction (|Bz|) of about 0.48, while region 1306 hasa small absolute value of magnetic field component in the z-direction(|Bz|) around 0.

FIG. 13B depicts 5×5 arrays 1330 and 1350 with a separation distance1372 of 1.4 mm. In contrast to FIG. 13A, magnets of arrays 1330 and 1350have their magnetic dipole moments pointing in the same directionwithout an alternating arrangement. Image 1362 shows the calculatedabsolute value of magnetic field component in the z-direction (|Bz|) forregion 1361 between the arrays 1330 and 1350. z=0 mm corresponds to thebottom surface of array 1330 and z=1.4 mm correspond to the top surfaceof array 1350. Most of the region 1361 has absolute value of magneticfield component in the z-direction (|Bz|) of about 0.24 and isrelatively uniform compared to the case of region 1301 in FIG. 13A.Region 1366 has a small absolute value of magnetic field component inthe z-direction (|Bz|) around 0. Thus, comparing to FIG. 13A, theconfiguration shown in FIG. 13B, has a relatively uniform absolute valueof magnetic field component in the z-direction (1134) and with a smallerpeak value.

To compare the above two configurations, FIG. 14 is a plot 1400presenting the calculated magnitude of magnetic field (|{right arrowover (B)}|) along cross-section lines 1314 and 1374 of FIGS. 13A and13B. Solid curve 1402 is the magnitude of magnetic field (|{right arrowover (B)}|) along cross-section line 1314. Dashed curve 1404 is themagnitude of magnetic field (|{right arrow over (B)}|) alongcross-section line 1374. The solid curve 1402 has an average magnitudeof magnetic field (|{right arrow over (B)}|) of about 0.35 T. In someembodiments, the average magnitude of magnetic field (|{right arrow over(B)}|) of solid curve 1402 can be about 0.3 or more (e.g., between 0.3 Tand 0.35 T, between 0.35 T and 0.4 T, between 0.45 T and 0.5 T).Moreover, the solid curve 1402 has a larger peak value of about 0.47 Tand larger gradient than that of dashed curve 1404. Accordingly, theconfiguration of arrays 1329 and 1349 can provide a stronger magneticforce according to Eq. (1) than the configuration of arrays 1330 and1350. In some embodiments, the peak magnitude value of solid curve 1402can be about 0.45 or more (e.g., between 0.45 T and 0.5 T, between 0.5 Tand 0.55 T, or between 0.55 T and 0.6 T).

FIG. 15 is a plot 1500 presenting the calculated magnitude of magneticforce |{right arrow over (F)}_(B)| of a spherical particle with a 1 μmradius and susceptibility χ=1 along cross-section lines 1314 and 1374.Solid curve 1502 is the calculated magnitude of magnetic force |{rightarrow over (F)}_(B)| along cross-section line 1314. Dashed curve 1504 isthe calculated magnitude of magnetic force |{right arrow over (F)}_(B)|along cross-section line 1374. As shown, the values of solid curve 1502are about 100 times larger than that of dashed curve 1504. Accordingly,plot 1500 shows that the magnetic force in region 1301 between arrays1329 and 1349 with alternating magnetic dipole moments can besignificantly larger that the case of region 1361 between arrays 1330and 1350.

Example 2—Testing of Particle Isolation

To test the efficiency of arrays of magnets 102 and 104, an experimentwas carried out to measure enrichment ratios depending of flow rates ofa sample fluid. FIG. 16 is a schematic diagram of a portion of amicrofluidic device 1610 used in the measurement. In a manner similarlydescribed in relation to FIG. 5A, arrays of magnets 102 and 104 (notshown) sandwiched the channels 506, which had a width of about 1 mm. Thesample fluid including non-magnetic particles 1629 and green magneticparticles 1630 were inserted into inlet 502. The sample fluid flowedthrough channels 506 along channel “1,” “2” and “3” according todirections 1601. Image 1602 is a zoomed view of a region includingchannels labeled “1,” “2” and “3.” Most of the green magnetic particles1630 were and attracted and stuck to walls of channel “1” by themagnetic fields provided by arrays of magnets 102 and 104. This resultshows that the arrays of magnets 102 and 104 can be effective incapturing magnetic particles 1630 without the need of passing multiplewindings of channel 506.

FIG. 17 is a plot 1700 showing measurement results of enrichment ratiosrelating to the number of non-magnetic particles 1629 and magneticparticles 1630 before and after passing through channel 506. The numberof non-magnetic particles (P1) and the number of magnetic particles (M1)before passing the channels 506 were measured using a flow cytometer(LSRII, BD Biosciences). The number of non-magnetic particles (P2) andthe number of magnetic particles (M2) after passing the channels 506were also measured. The enrichment ratio was calculated according to:

$\begin{matrix}{{{enrichment}\mspace{14mu} {ratio}} = {\left( \frac{P\; 2}{M\; 2} \right)/{\left( \frac{P\; 1}{M\; 1} \right).}}} & (2)\end{matrix}$

Bars 1702, 1704 and 1706 are the calculated enrichment ratio at flowrates of 2 mL/hr, 5 mL/hr and 20 mL/hr, respectively. In particular, theuse of arrays of magnets 102 and 104 achieved a high enrichment ratio ofabout 3500 at a high flow rate of 20 mL/hr.

During the measurements, an image of inlet 502 showed a mixture of thenon-magnetic particles 1629 and the green magnetic particles 1630. Onthe other hand, an image of outlet 504 showed only the non-magneticparticles 1629. These results demonstrated that a majority of the greenmagnetic particles 1630 were isolated by the arrays of magnets 102 and104 and the non-magnetic particles 1629 dominantly reached the outlet504.

The experiment also demonstrated trapping and displacing a particleusing an optical tweezer device. FIG. 18A is an image 1810 showing aparticle capture site 1802 in the microfluidic device 1610. A singlenon-magnetic particle 1629 is captured in the particle capture site1802. In this example, non-magnetic particle 1629 is not bound to amagnetic bead. The optical tweezer generated an optical trap 1806.

FIG. 18B is an image 1820 showing the optical trap 1806 trapping thenon-magnetic particle 1829. By moving the focus position of the opticaltrap 1806 in a direction out of the drawing plane, the trappednon-magnetic particle 1629 was lifted upwards of the particle capturesite 1802. Then the optical trap 1806 released the lifted non-magneticparticle 1829. FIG. 18C is an image 1830 showing the releasenon-magnetic particle 1629 being displaced by fluid flowing in direction1840. The results demonstrated that the optical tweezer could manipulateand release a selected particle trapped in a particle capture site.

FIG. 19 is an image 1900 showing a particle capture zone of anotherexperiment. In this experiment, following a depletion of host bloodcells, target cancer cells were captured at the particle capture zone.The pink circular shapes of regions 1904 and 1906 were stained targetcancer cells. A plurality of particle capture sites 1902 was designed totrap cancer cell of 5 μm or more in diameter. The particle capture sites1902 had underpass-gaps at the middle of capturing sites to enhancecapturing efficiency and remove red blood cells. Region 1904 correspondsto particle capture sites 1902 which captured single cancer cells.Region 1906 corresponds to particle capture sites 1902 which capturedtwo or more cancer cells. As shown in image 1900, the particle capturesites 1902 had higher probability of capturing a single cancer cell thanmultiple cancer cells.

Example 3—Lymphoma Detection

Implementations of the system 100 can be used for a variety ofexperimental and clinical applications. To demonstrate one example of anapplication, an experiment was carried out to investigate cerebralnervous system (CNS) lymphoma in cerebrospinal fluid.

A microfluidic device 110 was fabricated to meet several exemplarycriteria for processing CSF samples, including 1) a large number(e.g., >20,000) of cell capture sites to increase the likelihood ofidentifying monoclonal population when lymphoma cells make up as few as0.1% of the total cells; 2) antibody-free and sized-based capturestructure for cells in the 8-12 μm size range; and 3) pass-through gapsto remove erythrocytes.

Device Fabrication

In this experiment, the 2×4 cm² microfluidic device contained 24,000staggered, butterfly-shaped traps arranged in four bands of 20×300. Thefluidic system had a single-layer structure that is composed of acapture site region, a fluidic channel, and a debris filter at theinlet. Injected fluids (e.g. cells, buffers, antibodies) first passthrough the microfilter array (200 μm in diameter) in order to filterlarge aggregates and debris. The fluids then passed through the capturesite region (12000 μm in width; 5800 μm in length). FIG. 20 shows thedetailed dimensions of the single-cell capture sites 2002, which weredesigned to capture lymphocytes ˜10 μm in diameter. The microfluidicdevice includes two capture zone with different gap sizes (W1=30 μm and16 μm; L2=40 μm and 25 μm) for enhancing the capture rate. The height ofthe fluidic channel is 25 μm.

The capture site architecture was optimized to trap a single lymphocyte,while a 4-μm gap between the butterfly “wings” was incorporated to allowsmaller cells, such as erythrocytes, to pass through without beingcaptured. The microfluidic devices were fabricated via standard softlithography. In brief, an epoxy-based photoresist (SU-8 2025, MicroChem)was used to pattern a microfluidic channel on a silicon wafer. The waferwas then treated with trichlorosilane (Sigma Aldrich) under vacuum (1hour).

Polydimethylsiloxane (PDMS, Dow Corning) pre-polymer was mixed with acuring agent at a ratio of 10:1 (w/w), degassed under vacuum, and pouredover the channel mold. The polymer was then cured on a hotplate (60° C.,1 hour). The cured PDMS structure was then peeled off, treated with O₂plasma, and irreversibly bonded to a glass slide. Before use, eachdevice was flushed with pluoronic copolymer solution (0.02 wt % F127 inwater).

Sample Preparation

The cells used in this experiment were acquired from the followingsources: DB, Toledo (Dr. Anthony Letai, Dana Farber Cancer Institute);RC-K8 (Dr. Thomas Gilmore, Boston University); SuDHL4, DOHH-2, Rec-1(Dr. Russell Ryan, Massachusetts General Hospital); Daudi, Hut-78,Jurkat (ATCC). All cell lines (except Hut-78) were cultured (37° C. and5% CO₂) in RPMI 1640 media (Invitrogen) supplemented with 10% fetalbovine serum (FBS). Hut-78 cell line was cultured (37° C. and 5% CO₂) inIscove's Modified Dulbecco's Medium (Invitrogen) supplemented with 10%FBS.

The cells were titrated in this experiment as follows. Approximately1.5×10⁶ cells from culture flasks were washed with PBS and stained for30 min at room temperature in 1.5 μg/mL Hoechst 33342 (Invitrogen) andAPC anti-human-CD45 antibody according to manufacturer instructions(Clone HI30, BioLegend) in PBS containing 2% bovine serum albumin (BSA;Sigma Aldrich). Following a quick wash with PBS, cells were fixed in2.6% paraformaldehyde (PFA) in PBS at room temperature for 20 min. Cellswere then triple washed with PBS and counted using a hemocytometer(Hausser Scientific). The samples were diluted into quadruplicatealiquots of 10, 100, and 1,000 cells in 1 mL PBS in siliconizedmicrotubes (Clear-view Snap-Cap, Sigma-Aldich). Each sample was thenintroduced to a preconditioned device at a flow rate of 2 mL/hr. Thecaptured cells were then counted via microscopy.

Prior to processing by the system 100, each sample for prepared forimmuno-magnetic separation. In particular, given a sample of 5 mL, 5 mLPBS+4% BSA were added to each sample for final 2% BSA blocking at roomtemperature for 30 min. 5 mL were removed from each sample, and 25 uL ofanti-CD3-biotin (0.5 mg/mL, BioLegend 344820) were added per sample, andincubated for 60 minutes at 4° C. 100 uL streptavidin coated magneticbeads were then added per sample, and incubated for 30 minutes at roomtemperature. 25 uL of anti-CD19-PE (BioLegend) were added to eachsample, and incubated for 30 minutes at room temperature.

Experimental Methods

First, samples of cerebrospinal fluid were harvested, typically in therange of 1-3 mL. The collected samples were prepared for immune-magneticseparation (as described above), and immuno-magnetically separated usingthe system 100 in order to “pre-enrich” the sample prior to analysis,such that the relative abundance of lymphoma cells was increased. Eachsample was loaded onto the microfluidic device for immune-magneticseparation, whereby cells expressing CD3 (a T-cell marker) weremagnetically separated from cells that did not express CD3.

After separation, the remaining cells were captured in sub-nanolitertraps on the microfluidic device and stained within the microfluidicdevice for fluorescent imaging. Acquired images were then analyzed withan automatic computational technique to generate cell characterizationdata.

Staining on the microfluidic device was performed as follows. About 1000DB, Daudi, or a 1:1 mixture of cells were diluted into 1 mL ofartificial cerebrospinal perfusion fluid (aCSF; Harvard Apparatus).Samples were introduced to the device at the flow rate of 2 mL/hr.Following the cell capture, fix/perm buffer was perfused over the cellsfor 10 min, followed by permSB for 5 min, and PBS containing 2% FBS and1% BSA for 5 min, all at a flow rate of 1 mL/hr. A cocktail ofantibodies containing 1 μL of anti-Ki-67, anti-CD19, and anti-CD20, and2 μL of anti-κ light chain and anti-λ light chain was perfused over thecells at 1 mL/hr for 5 min. Lastly, to reduce background signal fromantibodies binding to the channel surface, washing buffer (PBS with 2%FBS and 1% BSA) was perfused at 1 mL/hr for 5 min. Alternatively, cellswere exposed to Ibrutinib-BFL using conditions recently described,followed by staining with Hoechst 33342 and anti-CD20-APC (clone 2H7;BioLegend). Images were captured on a Nikon Eclipse TE2000S invertedmicroscope (Nikon) equipped with four filter sets (#31000v2, #41001,#41002b, #41024; Chroma Technology).

Image analysis was performed as follows. Images were analyzed using anin-house Matlab (Mathworks) script. Briefly, images from the CD19/20(PE) channel were thresholded and binarized using Otsu's method.Following thresholding, image regions were analyzed and filtered byeliminating any regions greater or less then preset total pixel areasbased on the magnification of the images. Additional noise was filteredusing “open-close” morphological filtering. Boundaries of the remainingregions were then recorded and overlaid on target channels where valuesfor the pixels in each mask area for both lambda (Alexa Fluor 647) andkappa (Brilliant Violet 421) channels were generated. Final values forboth lambda and kappa channels for each cell were calculated byaveraging the most intense 25% of pixels in each region.

As the microfluidic device contains a large number of capturing sites,the microfluidic device allows for high-throughput analysis. Forinstance, with typical flow rates of 2-5 mL/hr, target cells could becaptured and stained in <1 hour, which may be of particular importancefor processing clinical samples.

The performance of the microfluidic device for cell capture wascharacterized. DB GCB-type DLBCL (diffuse large B-cell lymphoma) and theDaudi Burkitt lymphoma cell lines were stained for CD45 (anextracellular pan-lymphocyte marker) and nucleus, and samples wereprepared with the nominal cell counts of 10, 100, or 1000 of the DB orDaudi cells. When these samples were processed by the microfluidicdevice, the observed capture efficiency was >90%; this contrasts withthe 17-30% cell loss that occurs at each centrifugation step intraditional sample processing.

Fluorescent 10-μm microbeads (Bangs Laboratory) were used to testcapture efficiency and to find the optimal flow rate. The bead solutionwas diluted to a concentration of 3000 beads/mL, and an estimated 300beads were introduced to the device. Using a syringe pump, we appliednegative pressure at the channel outlet to generate fluidic flow. Thenumber of captured beads was counted via florescence microscopy. Theoptimal flow rate for maximal capture yield was determined to be between2-5 mL/hr. At lower flow rates, cells could have more time to follow thefluidic stream, thereby bypassing capture sites.

Captured cells were analyzed on the microfluidic device throughmulti-color immuno-microscopy. Three classifications were performed: 1)the use of CD19 and/or CD20 to determine B cells; 2) the use of kappa orlambda light chains to identify clonal populations; and 3) additionalphenotypic markers for subtyping and prognostic tasks. These markers andtheir respective antibodies were validated by profiling a panel of celllines via flow cytometry. Besides the B-cell lymphoma lines Daudi andDB, SuDHL4, DOHH2, and Toledo GCB-type DLBCL lines, the RC-K8 ABC-typeDLBCL line, and the Rec-1 mantle cell lymphoma line were also profiled.Hut-78, a T-cell line, was used as a control.

Results

The profiling results showed the importance of including both CD19 andCD20 to identify B cells; not all B-cell lines were found to expressboth markers. This finding is also supported by other reports thatshowed decreased CD20 in lymphomas either due to the cancercell-of-origin or anti-CD20 immunotherapy. The assay also showed therestricted expression of kappa or lambda light chain surfaceimmunoglobulins, which are markers of clonality, across the cell lines.

Several different lymphomas arise from germinal center B cells, such asBurkitt and some DLBCLs (GCB-type), but most primary CNS lymphomas areABC-type DLBCLs. As expected, we found that the GCB marker CD10 isexpressed in all the GCB cell lines tested, but not in ABC-type ormantle cell lymphoma. Since ABC-DLBCLs tend to be more aggressive, wechose Ki-67 as an important marker for characterization and prognosis.Our data for the DB, DOHH2, and Rec-1 lines suggests that low Ki-67 in amonoclonal population would indicate the need to test additionallymphoma markers, such as for GCB-type DLBCL or mantle cell lymphoma.MUM1 may also be important, as it was shown to be expressed in over 90%of PCNSLs.

Daudi and DB cells were used as a model system for analysis on themicrofluidic device, since they respectively highly express kappa andlambda light chain. To demonstrate both extracellular and intracellularantigen analysis, we performed staining of CD19/CD20, kappa/lambda, andKi-67 on the microfluidic device. We prepared cells for testing on themicro fluidic device by diluting DB and Daudi lymphoma cells intoartificial CSF. The cells were then fixed and stained on themicrofluidic device, and imaged in four channels. FIG. 21A shows anoverlay 2100 of the four imaging channels after a 1:1 mixture of DB andDaudi cells was captured and stained on the microfluidic device. Asshown in FIG. 21A, some cell capture sites 2104 exhibit fluorescence,while others do not. As several cell capture sites 2104 are shown in asingle image, the cells contained in each of these cell capture sites2104 can be analyzed from a single image. FIG. 21B shows two selectedportions of the overlay 2100 (a first portion shown in the row of images2106 a, and a second portion shown in the row of images 2106 b). Row2106 a corresponds to a first capture site containing a Daudi cell, andeach of the images 2102 a-e of row 2106 a shows the first capture siteaccording to different fluorescent channels. Row 2106 b corresponds to asecond capture site containing a DB cell, and each of the images 2102f-j of row 2106 b shows the second capture site according to thedifferent fluorescent channels. In observing the presence or absence ofcertain types of fluorescence, one can identify differences in theexpression of particle biomarkers between the cells. For example, asshown in images 2102 b-c, the Daudi cell captured in the first capturesite expresses kappa, but does not express lambda. In contrast, as shownimages 2102 g-h, the DB cell captured in the second capture site doesnot express kappa, but expresses lambda. Thus, this experimentdemonstrates that that high-resolution imaging can be performed forindividual cells and their markers. Further, the experimental alsodemonstrates that although the cell populations appear to beheterogeneous, their restricted kappa/lambda expression can be seen athigher magnification.

As a proof-of-concept of lymphocyte analysis from clinical samples, wedeveloped an image-processing technique for clonality assessment.Following the staining of cells, we first made a mask around cellsexpressing CD19 and/or CD20, and then quantified the mean fluorescenceintensity from our target channels in each individual cell. The mask wasgenerated by performing intensity-based thresholding on each image, andfiltering the thresholded image to obtain binary masks. For example,FIG. 22A shows an image 2202 that depicts the intensity of light withinthe PE channel for a portion of the particle capture zone. FIG. 22Bshows an image 2204 that results after performing an intensity-basedthresholding on the image 2202 (e.g., where pixels greater than or equala particular intensity are identified as exhibiting fluorescence, whilepixels less than that particular intensity are identified as notexhibiting fluorescence). FIG. 22C shows an image 2206 of the resultingmasks 2210 that are generated based on the thresholded image 2204. Asshown in FIG. 22D, each of the generated masks 2210 were used to analyzeadditional images 2208 that depicts the intensity of light within adifferent color channel (e.g., color channels associated with otherfluorophores, such as Alexa Fluor 647 and Brilliant Violet 421). A sizefilter was also included to exclude non-cell debris from analysis (e.g.,debris 2212 shown in FIG. 22A).

As the masks 2210 were created based on the fluorescence associated withCD19 and/or CD20, the mask corresponds to the location of cellsexpressing CD19 and/or CD20 (e.g., lymphocyte cells). Thus, the mask canbe used to identify specific portions of the image known to containlymphocytes. These masks can be applied to other images (e.g., imagesdepicting fluorescent associated with Alexa Fluor 647 and BrilliantViolet 421 fluorescence to determine if the lymphocytes in those regainsare lambda or kappa-expressing cells.

FIG. 23 shows plots 2302, 2304, and 2306 depicting the distribution ofmasked regions of images of the capture zone on the basis of their AlexaFluor 647 intensity (lambda) and Brilliant Violet 421 intensity (kappa),analyzed according to the masked regions 2210. As shown in FIG. 23,after quantification, we were able to distinguish DB (lambda-expressing)cell populations (as shown in plot 2304) and Daudi (kappa-expressing)cell populations (as shown in plot 2302) from samples containing about1,000 cells. The combined distribution for both of the cell populationsare shown in plot 2306. Thus, we were able to differentiate between twodifferent types of cells within the sample population. We furtherperformed drug sensitivity testing that would be clinically useful toguide intrathecal and/or systemic chemo- and targeted therapies. We useda companion imaging drug that has recently been reported, Ibrutinib-BFL,which is an inhibitor of Bruton's Tyrosine Kinase (BTK); other imagingdrugs include fluorescent rituximab or caged methotrexate. Ibrutinib isapproved for several B-cell malignancies, including mantle celllymphoma, and the Rec-1 cell line has been shown to be sensitive to thedrug. FIG. 24 shows images 2402 of Rec-1 cells and Jurkat cells withIbrutinib-BFL on the microfluidic device. As shown in FIG. 24, Rec-1cells exhibit fluorescence when incubated with Ibrutinib-BFL, andsimilarly exhibit fluorescent when incubated with an anti-CD20fluorescent marker. However, Jurkat cells do not exhibit fluorescentwhen incubated with either Ibrutinib-BFL or the anti-CD20. Thus, imagingthe Rec-1 cells with Ibrutinib-BFL on the microfluidic device shows notonly the binding of Ibrutinib, but also their cell-to-cell heterogeneitydue to differences in BTK inhibitor sensitivity and BTK proteinturnover. As such, this demonstrates that implementations of the system100 can be used to differentiate between cell types (e.g., CD20expressing cells vs. non-CD20 cells) as well as between cell types thatare differently sensitive to certain types of drugs (e.g., Ibrutinib).

CNS lymphoma is often difficult to diagnose and characterize at the siteof disease, in many cases requires multiple invasive lumbar punctures toretrieve sufficient numbers of cells to allow for cytopathologicanalysis. This experiment demonstrates that the paucicellularity andheterogeneity of CSF samples can be overcome through implementations ofthe above described microfluidic system that enable characterization ofpopulations of lymphoma cells in the CSF on a single-cell level. Here,we show that we can indeed image both intracellular and extracellulardiagnostic markers from lymphoma cells spiked into artificial CSF inunder an hour, and further use an image-processing algorithm toquantitate their expression level. By adding additional criteria,differential diagnosis using the Hans algorithm can identify thecell-of-origin of a PCNSL, perhaps pointing to an undiagnosed systemiclymphoma if germinal center origin is found. Thus, this experimentdemonstrated that the system 100 can be used to analyze different typesof cells on the basis of their intracellular and extracellularbiomarkers, and demonstrated that this can be used to different betweendifferent types of disease cell subtypes. Further, this experiment alsodemonstrated that the system 100 can be used to examine time-dependentcharacteristics of one or more cell types.

For secondary CNS lymphoma, it is often important to know the extent ofdisease, its aggression, and its response to treatment. Methotrexate iscurrently used intrathecally or at very high systemic doses to treat CNSdisease, but it has thus far not been possible to track response totreatment other than by low resolution MRI or insensitive cytology,neither of which would catch minimal disease. The system 100 enablesprofiling lymphoid cells in CSF based on kappa/lambda restriction orproliferative grade, or customizing antibody staining for intracellularor extracellular markers based on particular characteristics of theprimary tumor (e.g. c-Myc rearrangement, CD10, CD5, etc.) in order totrack CNS lymphoma cell counts c over time and to make prognosticassessments. Additionally, there are several new lymphoma drugs inclinical trials, yet few are tested for CNS efficacy. The system 100 canalso provide a companion diagnostic that can directly test forbrain-blood-barrier drug permeability or look for specific markerinhibition following intrathecal administration, such as BTK inhibitorsor new generation anti-CD20 agents. For example, as demonstrate above,to test for such drug accumulation (single cell pharmacokinetics) inprimary lymphoma cells, we tested Ibrutinib-BFL directly on amicrofluidic device. Finally, the system 100 also allows for the removalof CSF after intrathecal injection of chemotherapy drugs to tracktreatment response over time.

Although the above described experiment demonstrates one application ofthe described implementations, this is merely an illustrative example.In practice, implementations can be used for different applications thanthose described above. Further, the techniques used in the experimentcan be modified to suit the application at hand. As an example, in somecases, B cells can be further purified by negative selection of othercell types, such as T cells and monocytes. Since the capture is passive(e.g., no antibodies are used to capture cells within the capture zoneof the microfluidic device), in some cases, we can also use tweezingtechniques to remove single cells of interest off the microfluidicdevice for further characterization, such as by quantitative PCR andsequencing. In some cases, an imager or smartphone camera readout can beused to enable the microfluidic device to be used for lymphoma diagnosisin resource-poor settings. In some cases, a 1:5 cutoff ratio ofkappa-to-lambda fluorescence signal would be enough to establishclonality with high specificity.

OTHER EMBODIMENTS

It is to be understood that while various implementations have beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1.-36. (canceled)
 37. A system for isolating particles from a fluidsample, comprising: a first array of magnets arranged in a firsttwo-dimensional checkerboard pattern of magnets with directly adjacentmagnets in the first array having dipole moments aligned in oppositedirections; a second array of magnets arranged in a secondtwo-dimensional checkerboard pattern of magnets with directly adjacentmagnets in the second array having dipole moments aligned in oppositedirections; and a microfluidic device located between the first array ofmagnets and the second array of magnets, wherein the microfluidic devicecomprises: an inlet configured to receive the fluid sample; an outlet;and one or more channels extending between the inlet and the outlet,wherein at least a portion of at least one of the one or more channelsis positioned between the first array of magnets and the second array ofmagnets.
 38. The system of claim 37, wherein the second array of magnetsis arranged generally in parallel with the first array of magnets. 39.The system of claim 37, wherein the microfluidic device comprises aparticle capture zone in fluid communication with the one more channels,wherein the particle capture zone comprises a plurality of particlecapture sites.
 40. The system of claim 39, wherein at least one of theparticle capture sites comprises: a receptacle sized to confine a firsttype of particle; and an opening in fluid communication with the outlet.41. The system of claim 40, wherein the size of the receptacle is largerthan a diameter of the first type of particle.
 42. The system of claim41, wherein the receptacle allows passage of particles smaller than theopening.
 43. The system of claim 37, further comprising: a tweezerdevice configured to displace a particle captured in one of the particlecapture sites; and a receiver device configured to receive the displacedparticle.
 44. The system of claim 43, wherein the tweezer devicecomprises an optical source for generating an optical beam, and a lensfor focusing the optical beam into one of the particle capture sites.45. The system of claim 37, wherein at least one particle capture sitecomprises a first wall and a corresponding second wall, wherein thereceptacle is bounded by the first wall and the second wall, and whereinthe opening is defined between an end of the first wall and an end ofthe second wall.
 46. The system of claim 37, further comprising a fluidmanifold positioned between the particle capture zone and the outlet.47. A method for isolating particles, the method comprising: providing afirst array of magnets and a second array of magnets, wherein the firstarray of magnets is arranged in a first two-dimensional checkerboardpattern of magnets with directly adjacent magnets in the first arrayhaving dipole moments aligned in opposite directions, and wherein thesecond array of magnets is arranged in a second two-dimensionalcheckerboard pattern of magnets with directly adjacent magnets in thesecond array having dipole moments aligned in opposite directions;providing a microfluidic device between the first array of magnets andthe second array of magnets, wherein the microfluidic device comprisesone or more channels, wherein at least a portion of at least one of theone or more channels is positioned between the first array of magnetsand the second array of magnets; and flowing a sample fluid comprising aplurality of particles through the one or more channels, wherein atleast one first type of particle is bound to a magnetic bead, andwherein a magnetic field extending between the first array of magnetsand the second array of magnets within the one or more channels causesthe at least one first type of particle to separate from remainingparticles in the fluid sample.
 48. The method of claim 47, furthercomprising: flowing the fluid sample containing the remaining particlesto a particle capture zone of the microfluidic device, wherein theparticle capture zone comprises a plurality of particle capture sites.49. The method of claim 48, wherein at least one of the particle capturesites comprises a receptacle that is larger than a diameter of a secondtype of particle in the remaining particles, and wherein the receptacleallows passage of other remaining particles smaller than the opening.50. The method of claim 49, further comprising: capturing one or more ofthe second type of particles in at least one of the receptacles of theparticle capture sites of the microfluidic device, wherein the one ormore second type of particles are not bound to a magnetic bead.
 51. Themethod of claim 50, further comprising: displacing the one or moresecond type of particles from the particle capture sites using anoptical tweezer.
 52. The method of claim 51, wherein displacing the oneor more second type of particles from the particle capture sitescomprises displacing a single one of the second type of particles from asingle one of the particle capture sites.
 53. The method of claim 51,further comprising: flowing a first additional fluid through at leastthe particle capture zone, wherein the first additional fluid comprisesa plurality of first fluorescent markers; and allowing the plurality ofthe first fluorescent markers to bind to one or more of the second typeof particle.
 54. The method of claim 53, further comprising: opticallyexciting the first fluorescent markers bound to the one or more secondtype of particle; obtaining a first image of the one or more second typeof particle; and determining a characteristic of the second type ofparticle based on the obtained first image.
 55. The method of claim 54,further comprising: flowing an elutant through at least the particlecapture zone, wherein flowing the elutant causes the fluorescentlylabeled particles to release from the second type of particle; andflowing a second additional fluid sample through at least the particlecapture zone, wherein the second additional fluid comprises a pluralityof a second type of fluorescent marker, and wherein one of more of thesecond type of fluorescent markers bind to one or more of the secondtype of particle.
 56. The method of claim 55, further comprising:optically exciting one or more of the second fluorescent markers boundto the second type of particle; obtaining a second image of the secondtype of particle; and determining a characteristic of the second type ofparticle based on the obtained second image.